Abundance of Oligoflexales bacteria is associated with algal symbiont density, independent of thermal stress in Aiptasia anemones

Abstract Many multicellular organisms, such as humans, plants, and invertebrates, depend on symbioses with microbes for metabolic cooperation and exchange. Reef‐building corals, an ecologically important order of invertebrates, are particularly vulnerable to environmental stress in part because of their nutritive symbiosis with dinoflagellate algae, and yet also benefit from these and other microbial associations. While coral microbiomes remain difficult to study because of their complexity, the anemone Aiptasia is emerging as a simplified model. Research has demonstrated co‐occurrences between microbiome composition and the abundance and type of algal symbionts in cnidarians. However, whether these patterns are the result of general stress‐induced shifts or depletions of algal‐associated bacteria remains unclear. Our study aimed to distinguish the effect of changes in symbiont density and thermal stress on the microbiome of symbiotic Aiptasia strain CC7 by comparing them with aposymbiotic anemones, depleted of their native symbiont, Symbiodinium linucheae. Our analysis indicated that overall thermal stress had the greatest impact on disrupting the microbiome. We found that three bacterial classes made up most of the relative abundance (60%–85%) in all samples, but the rare microbiome fluctuated between symbiotic states and following thermal stress. We also observed that S. linucheae density correlated with abundance of Oligoflexales, suggesting these bacteria may be primary symbionts of the dinoflagellate algae. The findings of this study help expand knowledge on prospective multipartite symbioses in the cnidarian holobiont and how they respond to environmental disturbance.

Understanding the impact of multipartner associations on the health and survival of marine invertebrates is crucial given they are among the most susceptible animals to the impacts of climate change (Lam et al., 2020;Mather, 2013).The global decline of corals, which are cnidarian hosts that harbor intracellular populations of dinoflagellate algae in the family Symbiodiniaceae and other microbes (Pandolfi et al., 2003), is already underway and is predicted to worsen with climate change (Allemand & Osborn, 2019;Hoegh-Guldberg et al., 2007;Kleypas & Kleypas, 2019).Bleaching, which involves the expulsion of symbiotic algae in cnidarians resulting in the loss of color, can be induced by several factors, though elevated temperature, as noted by Douglas (2003), is the most common cause.Yet there is also abundant variation in coral thermal tolerance evidenced by differences in bleaching among species, populations, and individuals (Dixon et al., 2015;Drury, 2020;Thomas et al., 2018).Collectively, these observations have led to the "Coral Probiotic Hypothesis" (Reshef et al., 2006) which is based on the notion that symbiotic relationships with bacteria can increase coral resilience (Peixoto et al., 2017).
The cnidarian-dinoflagellate symbiosis is better characterized in the literature than potential cnidarian-bacterial symbioses, and it remains unclear whether environmental or internal host factors, like host genetics or microalgal symbiont type, modulate the composition of cnidarian microbiomes (Barno et al., 2021;Bourne et al., 2016;van Oppen & Blackall, 2019).One understudied theory postulates that well-known members of the cnidarian microbiome may actually be obligatory members of the Symbiodiniaceae microbiome, in both free-living and in hospite states (Bernasconi et al., 2019;Matthews et al., 2020;Ritchie, 2012).Global datasets suggest that the identity of Symbiodiniaceae may contribute to structuring coral and anemone microbiomes (Bernasconi et al., 2019).For instance, susceptibility to Vibrio pathogens was higher in Acropora cytherea corals hosting Symbiodinium than Durusdinium (formerly Clades A and D Symbiodinium, respectively) (Rouzé et al., 2016).Additionally, the abundance of diazotrophs in Montipora corals also correlated with algal symbiont type (Olson et al., 2009).Similarly, unique microbiomes were identified in symbiotic and aposymbiotic Aiptasia anemones (Herrera et al., 2017), indicating presence of the symbiont influences the microbiome.However, insufficient evidence exists to verify this hypothesis and tracking bacteria in adult corals is nearly impossible, due to their high bacterial diversity (Blackall et al., 2015), which poses a challenge for distinguishing obligatory and facultative bacterial symbionts.Furthermore, selectively eliminating holobiont members empirically is not feasible since reef-building corals cannot survive without their algal symbionts, who provide vital sugars and nutrients (Weis, 2008).The anemone Aiptasia (Exaiptasia pallida, sensu stricto; Grajales & Rodríguez, 2014) is a tractable model for studying the cnidarian microbiome as it harbors fewer bacterial taxa, with diversity estimated to be around 1-2 orders of magnitude lower than their coral relatives (Herrera et al., 2017;Röthig et al., 2016).
Aiptasia are easy to maintain, engage in a nutritive symbiosis with Symbiodiniaceae similar to corals, and can reproduce asexually (Baumgarten et al., 2015;Weis, 2019).Unlike their coral relatives, Aiptasia can be rendered aposymbiotic (free of their dinoflagellate algae) in laboratory conditions and lack a calcitic skeleton, which facilitates experimental manipulation (Lehnert et al., 2014).
While associations between microbial community composition and the abundance and/or diversity of algal endosymbionts is consistent with the hypothesis that some microbes are obligate symbionts of Symbiodiniaceae, these patterns cannot be distinguished from commensal relationships, where one partner benefits and the other remains unaffected.Additionally, general stress may be responsible for changes in the host-photosymbiont relationship, leading to alterations in metabolite production and resulting in different selective pressures that favor distinct groups of commensals.For example, Symbiodiniaceae taxa are known to differ in their metabolite production (Camp et al., 2022).Durusdinium translocates less carbon to hosts than Cladocopium (Cantin et al., 2009) under thermal stress, but Cladocopium translocates more carbon and nitrogen to hosts during nonstressful conditions (Pernice et al., 2015), which could impact the consortium of microbiota in the holobiont.The host-photosymbiont relationship can also be affected by mild bleaching, which is another general stress response (Ortiz et al., 2009).Here, we aimed to distinguish the effects of the stress response and that of symbiont density on microbial communities while controlling for host and symbiont genetic diversity.To achieve this, we used the emerging model organism Aiptasia clonal strain CC7, which harbors Symbiodinium linucheae (Baumgarten et al., 2015;Starzak et al., 2014) and conducted a comparison between the microbiomes of symbiotic anemones undergoing mild bleaching and aposymbiotic anemones, which were presumed to lack the microbes typically associated with Symbiodinium.

| Aiptasia rearing
Aiptasia anemones, clone strain CC7 (obtained from Dr. Cory Krediet, Eckerd College, FL, USA), were used in this study.Anemones were kept in 0.5 L polycarbonate tanks, filled with 0.2 μm filtered seawater (FSW) from Catalina Island (Catalina Water Co., Long Beach, CA, USA), and maintained at 25°C on a light/dark (14:10 h) cycle under 12-20 μmol/m 2 /s.Animals were maintained in these common garden conditions at USC with weekly feeding (frozen brine shrimp, Artemia salina) and water changes for 24 months.A subset of Aiptasia animals were rendered aposymbiotic by menthol-induced bleaching (Matthews et al., 2016) 4 months prior to experimental trials.Briefly, a 1.28-M menthol solution was prepared (20% w/v in ethanol) and added to polycarbonate tanks containing FSW for a final concentration of 0.19 mmol/L.Anemones were placed in the menthol solution for 8 h during the light period of the 14:10, light:dark cycle.The animals were then transferred overnight to tanks containing a final concentration of 0.10 M DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylur ea), an algicide and photosynthesis inhibitor.This was repeated for four consecutive days.At the end of the fourth day, anemones were placed in blackout tanks with no light exposure and allowed to recover for 3 days with one feeding.After the 3-day recovery, the process was repeated once more and bleaching status was confirmed using a fluorescent microscope.An absence of red autofluorescence from dinoflagellate chloroplasts was noted and these aposymbiotic anemones were transferred to blackout tanks filled with 0.2 μm FSW and maintained in the dark for 3 months with the same feeding and water change regime as the symbiotic anemone stock.

| Experimental design and sampling
Symbiotic and aposymbiotic anemone of approximately 2.5 cm length were selected for the 6-day thermal stress experiment.
Aposymbiotic Aiptasia were added to the experimental design to act as a control for distinguishing baseline host stress responses from cnidarian-algal microbiome shifts.These aposymbiotic anemones were acclimated to the same light/dark (14:10 h) cycle as symbiotic anemones 1 week prior to the experiment start date.In addition, all anemones were starved for 2 weeks prior to sampling to avoid prey (shrimp, Artemia sp.) contamination.
A total of 18 symbiotic anemones (6 per 0.5 L tank, × 3 tanks) and 12 aposymbiotic anemones (6 per 0.5 L tank, × 2 tanks) were used per experimental condition (25°C, 32°C) (Figure 1).The treatment temperature was set to 32°C due to prior observation of slight bleaching and expulsion of Symbiodinium in Aiptasia at this temperature (Perez et al., 2001).To maintain and manipulate temperature, tanks used in the heat stress treatment were placed in 10 L bins containing two SL381 submersible water pumps, two 100 W aquarium heaters, a HOBO temperature logger, and a digital, waterproof thermometer.Acclimatization of Aiptasia was reached by exposing them to a gradual temperature ramp over the course of 4 days.Temperature was increased from 25 ± 0.5°C to 27 ± 0.5°C the first day to 30 ± 0.5°C the second day, 32 ± 0.5°C the third day, and 33 ± 0.5°C on the fourth day.Once Aiptasia were acclimated to the elevated temperature treatment, treatment was maintained at an average of 32 ± 0.5°C for 6 days with FSW changes every 2 days, in both control and heat stress tanks.At the end of the 6-day exposure period, each anemone was rinsed three times with FSW in individual 60 × 15 mm Petri dishes, placed in a 1.5-mL microcentrifuge tube, and frozen at −80°C, until processing.

F I G U R E 1
Study design for the mild thermal stress experiment.Each treatment condition received five tanks (three tanks × six symbiotic anemones and two tanks × six aposymbiotic anemones; n = 18 symbiotic anemones per treatment and n = 12 aposymbiotic anemones per treatment).Following a 7-day ramping period, the peak temperature exposure continued for 6 days, and sampling was conducted on the last day.Each anemone was rinsed with filtered seawater (FSW) thrice, deposited into a microcentrifuge tube, and stored at −80°C until processing.DNA extractions were performed followed by 16S rDNA amplicon sequencing.Samples with remaining DNA were used in qPCR assays to determine symbiont (Symbiodinium) to host (Aiptasia) ratios.

| DNA extractions
Individual anemone DNA was extracted by ethanol precipitation as detailed previously (https:// openw etware.org/ wiki/ Ethan ol_ preci pitat ion_ of_ nucle ic_ acids ) with some modifications.Briefly, animals were placed in sterile, 2 mL polypropylene screw cap tubes (Merck KGaA, Germany) containing a thin layer of Zi/Si beads (100-500 mm diameter), 200 μL lysis buffer AP1 from a DNEasy Power Plant Kit (Qiagen, Germany), 2 μL RNAse (100 mg/mL, stock), and 2 μL Proteinase K (20 mg/mL, stock) and incubated at 55°C for 10 min in a temperature-controlled water bath.Samples were then homogenized using the Omni bead beater (Omni International, USA) at 6.3 m/s for two cycles of 30 s.The samples were then centrifuged for 5 min at 14,000 rpm and the proteinase K enzyme was heat inactivated on a heat block (78-82°C) for 3 min.The supernatant was transferred to a 1.5-mL microcentrifuge tube.Ethanol precipitation was performed as described previously (https:// openw etware.org/ wiki/ Ethan ol_ preci pitat ion_ of_ nucle ic_ acids ) and the pellet was resuspended in 40 μL elution buffer (GenElute Bacterial Genomic DNA Kit, Merck KGaA, Germany).DNA was purified using the Zymo DNA Clean and Concentrator Kit (Zymo Research, USA) following the manufacturer's instructions.
Additional statistical analyses were conducted using the vegan (Dixon, 2003) package.A one-way ANOVA test was conducted on rarefied data, comparing alpha-diversity data (Chao1 scores) between the microbial assemblages of symbiotic and aposymbiotic anemone groups in different treatments (control vs. thermal stress).Post hoc pairwise comparisons were done using Tukey's HSD.Beta diversity was visualized using a PCoA plot employing the weighted Unifrac metric.The adonis2 function in vegan was used to conduct pairwise permutational multivariate analysis of variance (PERMANOVA) of microbial assemblage dissimilarities between treatment groups.
Adonis2 was used due to even homogeneity of variances in all pairwise comparisons tested.Tank effects were minimal to nonexistent (Figure S1b) as visualized in a PCoA plot (weighted Unifrac metric).
As the SILVA SSU v138.1 database only assigned some bacterial taxonomy to order, we conducted a separate phylogenetic analysis of ASVs classified in the order Oligoflexales.A Bioconda (Grüning et al., 2018) environment was used for this analysis.The filtered Phyloseq dataset was subsetted to include only Oligoflexales reads and resulting ASVs were then transferred to a fasta file and a standard NCBI nucleotide blast (Sayers et al., 2023) was performed on the web interface, optimizing for highly similar sequences (megablast).The ASVs exhibited high-quality (Table S2) matches to three uncultured bacterium clones, originating from one microbial survey (Randle et al., 2020) conducted on Aiptasia strain CC7 (GenBank: MK571601.1/MK571569.1)and Aiptasia strain H2 (GenBank: MK571216.1),as well as one uncultured proteobacterium clone (GenBank: FJ425635) found in the microbiome of scleractinian coral Orbicella (formerly Montastraea) faveolata.We queried our nine Oligoflexales ASVs and these previously published sequences with 16S rDNA sequences from two confirmed members of the Oligoflexales order (GenBank accession numbers AB540021.2and OW948931.1)and 10 members (7 families) of the Bdellovibrionota phylum.Sequence alignment was performed using the MUSCLE algorithm version 5.1 (Edgar, 2004) and a phylogenetic tree was constructed by maximum likelihood with ultrafast bootstrap (n = 1000 replicates) in IQ-TREE version 2.2.0.3 (Kalyaanamoorthy et al., 2017;Minh et al., 2020).The resulting phylogenetic tree was visualized and annotated using the integrated web editor interface, Interactive Tree of Life (ITOL, https:// itol.embl.de/ ).

| Symbiont-to-host (S/H) cell ratio qPCR
To assess the effects of thermal stress on symbiont density in Aiptasia, we analyzed symbiont-to-host cell ratios (Mieog et al., 2009) using nuclear ribosomal protein L10 as a reference gene for the host, Aiptasia (Poole et al., 2016), and the actin locus as a target in Symbiodinium (Palacio-Castro, 2019).Nuclear ribosomal protein L10 primers were previously validated for Aiptasia specificity in qPCR assays (Poole et al., 2016) and used as a reference gene due to stable expression in Aiptasia (Kitchen & Weis, 2017) S3) for the qPCR assays.Each sample was assayed in duplicate, per target primer set.
Cycle threshold (C t ) values were calculated by Agilent AriaMx qPCR machine when the first amplification cycle in a reaction exceeded the fluorescent baseline.All aposymbiotic anemones in the control (but not those in the thermally stressed samples) exhibited nontarget amplification for the actin primer set, and upon analysis of the melt curve, we noticed a distinct peak between 83 and 84°C for these samples, yet all other positive amplification reactions exhibited a distinct peak between 85 and 87°C.We surmised crossamplification of an Aiptasia locus in the absence of a Symbiodinium target.Sanger sequencing of the different products revealed that actin samples with a melt product between 85 and 86°C exhibited high-quality matches to the actin gene locus in Symbiodinium spp.

| S/H ratio statistical analyses
A dataset containing only symbiotic Aiptasia with S/H ratios, their alpha-diversity scores (Shannon, Chao, Observed, Fisher and Simpson), and select bacterial abundance counts (Oligoflexales and Staphylococcus) was used to generate a Pearson's correlation matrix using the corrplot package in R (Wei & Simko, 2021).The lm command was used for regression analysis of Oligoflexales abundance counts on S/H ratio in symbiotic anemones.We implemented a linear mixed effects model with a fixed effect of treatment and a random effect of tank to test whether S/H ratios were reduced in symbiotic anemones response to heat treatment using the nlme (Bates & Pinheiro, 1998;Pinheiro & Bates, 2023) and lme4 (Bates et al., 2015) packages.

| Microbial assemblages in Aiptasia
After ASV calling with DADA2, the sequence table yielded 4,996,356 reads and after chloroplast and mitochondria removal, 4,984,116 reads and 4471 ASVs remained (Table S1).Rarefaction and filtering of taxa occurring at least three times in more than four samples (the minimum number of replicates per tank) yielded a final dataset of 1,338,082 reads and 774 ASVs.The dataset was dominated by Alphaproteobacteria (66%), Gammaproteobacteria (19%), and Bdellovibrionota (4%) (Figure 2, Figure S1a).All other taxonomic classes were present in abundances <3%, except for Oligoflexia, which occurred at 4% relative abundance in symbiotic anemones under control conditions (Figure S1a).The predominant genera in all samples were Cognatishimia, an unnamed bacterium from the family Rhodobacteraceae and Alcanivorax (Figure S2).

| Community dynamics differ by treatment and symbiotic state
Alpha diversity was assessed by estimating species richness using the Chao1 index.Lower within-sample diversity was observed in symbiotic anemones exposed to heat, but within-sample diversity was consistent between other treatment groups (Figure 3a).
Although alpha diversity differed between groups on average (ANOVA, p = .009,Table S5), significant differences were detected for only one pairwise comparison: heat-stressed symbiotic anemones and control symbiotic anemones (Tukey's multiple comparison of means, p = .016,Table S5).A marginal pairwise difference was detected between symbiotic and aposymbiotic anemones under heat stress (Tukey's multiple comparison of means, p = .05,

Table S5).
A principal coordinate analysis (PCoA, weighted Unifrac) also revealed differences in beta diversity between symbiotic and aposymbiotic pairs in response to treatment (Figure 3b).Namely, beta diversity in heat-stressed symbiotic anemones converged, whereas beta diversity in control symbiotic anemones did not (PERMANOVA, p = .001,Table S6), indicating microbial community composition in symbiotic anemones became more similar to each other after heat treatment.The opposite pattern was observed in aposymbiotic anemones: convergence was observed in the control group and divergence in the heat treatment (Figure 3b, PERMANOVA, p = .001,Table S6).Differences in beta diversity were also observed between symbiotic and aposymbiotic animals under control conditions (ANOSIM R = .28,p = .003,Table S6).
We further explored taxa responsible for beta-diversity disparities by generating a differential heat tree and visualizing statistically dissimilar taxa between pairwise comparisons.We detected differential abundance of several taxa (Wilcoxon rank sum tests, FDR-adjusted p values <.05; statistical output: Zenodo, https:// doi.org/ 10. 5281/ zenodo.7693398), but most noticeably Firmicutes, Oligoflexales, Oceanospirillales, Planctomycetes, and Alteromonadales (Figure 4).F I G U R E 4 Differential heat trees illustrating pairwise comparisons between groups of interest.Amplicon data were used to visualize microbial taxonomic composition in Aiptasia, using the R package, Metacoder (Foster et al., 2017).The bigger tree with taxon labels on the lower left serves as a key for the smaller pairwise comparison trees surrounding it (a) aposymbiotic control versus aposymbiotic heat, (b) aposymbiotic control versus symbiotic control, (c) symbiotic heat stressed versus aposymbiotic heat stressed, and (d) symbiotic heat stressed versus symbiotic control.Taxon color (diverging scheme from pink to green) is represented by log 2 ratio of median proportions of reads observed by treatment group.Significantly differentially abundant taxa, determined by Wilcoxon rank sum tests followed by FDR correction, colored in pink are more prominent in the groups shown in the columns and those colored in green are more prominent in the groups shown on the rows, for example, Oligoflexales are significantly more abundant in symbiotic control (green) anemones than symbiotic heat stressed, but Myxococcota are enriched in symbiotic heat stressed (pink).Size of tree nodes corresponds to ASV richness, as denoted in the color and size key in the upper right.Statistical output of differential abundance analysis is archived at Zenodo, https:// doi.org/ 10. 5281/ zenodo.7693398.

| Oligoflexales order are associated with symbiotic state and are lost under thermal stress
Oligoflexales abundances were elevated in symbiotic anemones under control conditions but marginally elevated under heat treatment in aposymbiotic anemones relative to aposymbiotic controls (Figure 4a,b).Total abundance counts clearly displayed this categorical difference as well (Figure 5b).In addition to these categorical differences in Oligoflexales abundance by anemone symbiotic state and treatment (Figure S1, Figures 4 and 5b), we also observed quantitative differences in abundance as a function of algal symbiont density.Symbiont-to-host (S/H) cell ratios of symbiotic anemones decreased under thermal stress indicating mild bleaching (p = .047,Figure S3, Table S7).Total S/H ratios for aposymbiotic anemones averaged around 0, but values slightly increased in anemones exposed to thermal stress (Figure S3, Table S4).
A correlation matrix was built to examine pairwise relationships between the S/H ratio of symbiotic Aiptasia, alpha diversity, and Oligoflexales abundance counts.A moderate correlation between S/H ratio and the abundance of Oligoflexales bacteria was detected (Pearson's correlation, p = .013,Figure S4, Table S8).Regression analysis verified this positive correlation, revealing that 53% of the observed variation in Oligoflexales abundance across samples was explained by differences in S/H ratio (p < .001, Figure 5a).

| Oligoflexales may diversify under thermal stress
A phylogenetic tree plotting Oligoflexales ASV abundance showed symbiotic anemones in control conditions initially hosted nine distinct ASVs but lost one after thermal stress (Figure S5).In contrast, aposymbiotic anemones only harbored two ASVs under control conditions, whereas eight Oligoflexales ASVs were detected in aposymbiotic anemones which experienced thermal stress (Figure S5).We conducted a more detailed phylogenetic analysis to further investigate relationships among these distinct ASVs and other Oligoflexales variants identified in prior studies.Oligoflexales ASVs from this study grouped closely with three uncultured bacterium clones originating from an unrelated study on Aiptasia clonal strains CC7 and H2 (Randle et al., 2020) and one uncultured proteobacterium clone (GenBank: FJ425635) found in the microbiome of the scleractinian coral Orbicella (formerly Montastraea) faveolata (Figure S6).Other Oligoflexales representatives were more distantly related (Figure S6).

| DISCUSS ION
Overall, understanding the ecological dynamics of algal-microbe interactions in cnidarians may be important for developing strategies to mitigate the impacts of climate change (Matthews et al., 2020).Frommlet et al. (2015) found that a diverse community of bacteria facilitated the formation of symbiolites (spheroid, aragonite structures) in ex hospite Symbiodiniaceae cultures.This discovery highlighted a unique approach that could potentially aid in coral calcification within reefs.Furthermore, microbiome and algal symbiont co-occurrences may be a result of metabolic cooperation, as dinoflagellates may rely on necessary metabolites produced by bacteria and vice versa (Cruz-López & Maske, 2016;Grossman, 2016;Kurihara et al., 2013).Additional possible functional roles of bacteria associated with Symbiodiniaceae may also span DMSP production, enhancing iron bioavailability and sulfur cycling (Lawson et al., 2018).
Here, we investigated the ecology of microbial communities, associated with Aiptasia, and how they are affected by the presence of the algal symbiont and elevated temperatures.Despite treatment, Alphaproteobacteria and Gammaproteobacteria remained the predominant microbial taxa.Differences in beta diversity were observed between symbiotic and aposymbiotic animals under both control and thermal stress conditions, but greater community similarity was observed among microbial populations in both symbiotic and aposymbiotic anemones exposed to heat stress.Additionally, elevated temperature decreased species richness in symbiotic anemones.We also show that Oligoflexales bacteria are part of the rare microbiome in symbiotic anemones, but significantly decreased in abundance following thermal stress.The abundance of Oligoflexales was positively correlated with higher S/H cell ratio indicating symbiont density, rather than heat stress per se, impacted their abundance in Aiptasia.

| Exploring the cnidarian-algal-bacteria tripartite symbiosis, Oligoflexales as primary associates of Symbiodiniaceae
We examined the role of symbiotic algae in recruiting microbial taxa that may be specific to the microbiomes of symbiotic Aiptasia and identified two taxa (Oligoflexales and Methylophilaceae) that were significantly abundant in symbiotic Aiptasia only and decreased in relative abundance with symbiont loss both independent of and as a result of thermal stress (Figures 4b,d and 5).We focused on exploring Oligoflexales because these taxa were present in higher relative abundances and had been previously reported as associates of Aiptasia (Maire et al., 2021;Randle et al., 2020), whereas we could not find a consistent record of Methylophilaceae in symbiotic Aiptasia.Despite numerous surveys conducted on the Aiptasia microbiome (Ahmed et al., 2019;Costa et al., 2021;Hartman et al., 2020;Herrera et al., 2017;Röthig et al., 2016), Oligoflexales remained undetected until recent research reported their presence in Aiptasia strain CC7 (Randle et al., 2020) and in the microbiome of Aiptasia acontia in strains AIMS1, AIMS2, AIMS3, and AIMS4 (Maire et al., 2021).Two factors likely account for these results: (1) the utilization of identical sequencing primer sets across all three studies, which document the existence of Oligoflexales, including our own, and (2) the classification of taxonomy based on the most recent release of the SILVA database (v138, issued in 2019).We chose to use the 784F/1061R primer set because it captures global bacterial diversity while exhibiting low amplification of chloroplast and mitochondrial host DNA (Andersson et al., 2008;Bayer et al., 2013).Furthermore, as Oligoflexales were recently recognized as a novel order under the Bdellovibrionota phylum (Nakai et al., 2014;Waite et al., 2020), older databases may designate them as "unclassified/ uncultured bacterial clones."Based on our research and the prior studies, we conclude that Oligoflexales are a consistent component of the symbiotic microbiome in Aiptasia.
Bacteria belonging to the Oligoflexales order are Gram-negative, oligotrophic spirochaetes, and only one species, Oligoflexus tunisiensis, has been described and isolated (Nakai et al., 2014(Nakai et al., , 2016)).We conducted a phylogenetic analysis of nine Oligoflexales ASV sequences against O. tunisiensis and other members of the parent phylum, Bdellovibrionota (Waite et al., 2020).Our ASVs formed a sister clade to O. tunisiensis and Pseudobacteriovorax antillogorgiicola, an isolate from gorgonian corals in the family Pseudobacteriovoracacea (McCauley et al., 2015) (Figure S6).This suggests a close relationship between them.Currently, Pseudobacteriovoracacea are classified as Bdellovibrionales, but a proposal to reclassify them as Oligoflexales was submitted (Hahn et al., 2017), which is consistent with our findings grouping them with O. tunisiensis.

Although the ecological role of Oligoflexales in symbiotic
Aiptasia remains a mystery, it has previously been suggested to comprise a set of taxa that aid thermotolerance in high salinities (Randle et al., 2020).The genome sequence of O. tunisiensis also provides clues on possible metabolic capabilities in Oligoflexales.Nakai et al. (2016) observed an incomplete denitrification pathway in O. tunisiensis, which resulted in the conversion of nitrate/nitrite (NO 3 / NO 2 ) to nitrous oxide (N 2 O).Heterotrophic bacteria are known to recycle fixed nitrogen from the environment through denitrification, and those with a complete pathway can reduce fixed nitrogen to dinitrogen (N 2 ) gas (Knowles, 1982).Nitrogen recycling by the host, Aiptasia, regulates algal symbiotic density (Cui et al., 2019), but bacterially regulated nitrogen may play a role in maintenance of the cnidarian-algal symbiosis as well, since nitrogen cycling is a hallmark of reciprocal bacterial association in cnidarian holobionts (Knowlton & Rohwer, 2003;Peixoto et al., 2017).It is unclear if the Oligoflexales in this study possess a similar denitrification pathway, but the genomic evidence from O. tunisiensis suggests that further research is needed to investigate this possibility.
We observed a higher abundance of Oligoflexales ASVs in symbiotic Aiptasia under control conditions, compared to symbiotic animals in the heat stress treatment.In contrast, aposymbiotic Aiptasia in the control showed only one ASV but experienced an increase in diversity and abundance of ASVs in thermally stressed, aposymbiotic anemones (Figure S5).While superficially this observation initially appears to contradict the notion that Oligoflexales are associates of Symbiodiniaceae, we believe this pattern can be explained by the dynamics of symbiont maintenance and proliferation in Aiptasia (Jinkerson et al., 2022) and is actually fully consistent with the primary algal symbiont hypothesis.
Aposymbiotic Aiptasia anemones can retain remnant populations of algal symbionts, even in the absence of light.Symbiodinium linucheae do not need to photosynthesize to be maintained in Aiptasia in dark conditions (Jinkerson et al., 2022).Jinkerson et al. (2022) also found that S. linucheae did not proliferate in hospite in the dark, but algal cell density significantly increased after subsequent transition to light conditions.We theorize that a small, nondetectable (by PCR) population of algal symbionts remained in the aposymbiotic group, despite continuous darkness for 3 months.This S. linucheae population likely proliferated during the combined light and heat stress periods as confirmed by the slight increase in S/H ratios in this population (Figure S3, Table S4), in contrast to the aposymbiotic controls only exposed to light.A recent study showed significantly higher proliferation of S. linucheae in Aiptasia at 32°C, after 28 days compared to ambient temperatures (25°C), suggesting cell division rates initially increased in response to elevated temperature but then declined after 12 weeks of sustained thermal stress (Herrera et al., 2021).The slight increase in algal abundance in the aposymbiotic population subjected to thermal stress was concomitant with an increase and diversification of Oligoflexales (Figure 4b, Figure S5).While the opposite pattern (Oligoflexales loss) was observed in symbiotic Aiptasia exposed to heat stress.S/H ratios were lower (Table S4, Figure S3) in symbiotic anemones exposed to thermal stress, and significant differences were observed between treatments (Table S7), indicating symbiont loss.Thermal stress can promote bleaching in symbiotic cnidarians leading to dysbiosis (Weis, 2008;Wooldridge, 2009), but the mechanisms are not thoroughly established.One theory suggests dysbiosis may occur as a cascade effect begins with a decrease in the photosynthetic efficiency of the symbiont, followed by a reduction in ammonium assimilation by the host, leading to an increase in the available ammonium pool.This, in turn, stimulates algal growth, and eventually, the host becomes unable to keep up with the resulting internal metabolic shifts, leading to the expulsion of the symbionts (Cui et al., 2019;Rädecker et al., 2021).We hypothesize that heatstressed, aposymbiotic Aiptasia did not experience the same metabolic constraints and were able to adequately support growth of algal populations, leading to slightly higher symbiont abundance and increased abundance/diversity of Oligoflexales bacteria (Figure 5b, Figure S5).
Finally, it is a possibility that Oligoflexales observed in aposymbiotic anemones under heat conditions were environmentally acquired.We used 0.2 μm FSW to maintain our cultures, but previous research has demonstrated that O. tunisiensis is part of the 0.2 μm filtrate culturable fraction (Nakai et al., 2014).Although O. tunisiensis can reach up to 10 μm in length, and are between 0.4 and 0.8 μm wide, they can compress through 0.2 μm filter pores.
We do not have morphological data for the Oligoflexales in this study, so we cannot eliminate the possibility of environmental contamination.However, all animals were clonal propagates and were maintained using 0.2 μm FSW, originating from the same 20 L tank, yet displayed distinct microbiome profiles according to treatment (Figures 2 and 3).Furthermore, the disparity of Oligoflexales abundances between treatment groups (Figure 5b) indicates symbiotic state specificity in control conditions.Environmentally acquired or not, Oligoflexales may play an important role in the holobiont as endosymbiotic partners that only associate with cnidarians when microalgal symbionts are present, as rare members of the symbiotic microbiome.It is possible that Oligoflexales, like rare members of the coral core microbiome, are widespread in anemone tissue, particularly in close proximity to algal symbionts (Ainsworth et al., 2015) and future work should aim to fully characterize their abundance and distribution.

| Microbiome assemblages of aposymbiotic and symbiotic Aiptasia
Microbial assemblages in Aiptasia from wild and cultured populations (including clonal strain CC7) in ambient conditions showed consistent similarity at the phylum level but not at lower taxonomic levels (Brown et al., 2017).In all of our samples, regardless of treatment, Alcanivorax sp. from the Oceanospirillales order, Cognatishimia sp., and an unclassified bacterium from the Rhodobacteraceae family from the Rhodobacterales order accounted for around 60%-85% of the total relative abundance (Figure S2).This observation, that 2-3 taxa are numerically dominant in Aiptasia, contradicts other microbial surveys on Aiptasia that identified a wider range of taxonomic groups within the same relative abundance range (Curtis et al., 2023;Randle et al., 2020;Röthig et al., 2016).The importance of functional redundancy in shaping microbial communities in Aiptasia is emphasized by the conflicting results obtained from various studies.Functional redundancy refers to a diverse range of bacteria with similar capabilities, able to perform similar functions in the same niche (Louca et al., 2018).Functional redundancy is an advantageous strategy for ecosystem stability and may play a role in the resilience of hosts, like corals, facing environmental disturbances (Voolstra & Ziegler, 2020).
Prior work (Ahmed et al., 2019;Randle et al., 2020;Röthig et al., 2016) showed that aposymbiotic and symbiotic Aiptasia (CC7) hosted distinct microbiomes.Here we expanded upon this finding by introducing a stressor to assess the response of the aposymbiotic microbiome and the symbiotic microbiome under thermal stress (Figure S1).Although alpha diversity did not differ between symbiotic status (Figure 3a), beta diversity was dissimilar (Figure 3b, Table S6), which prompted us to explore which taxa were responsible for these observations.We observed a significant difference in the relative abundance of Oligoflexales, Saccharospirillaceae, Pseudoalteromonadaceae, and Methylophilaceae families between symbiotic and aposymbiotic animals, with these taxa being more prevalent in the former (Figure 4b).Our findings align with other studies that have demonstrated differences in relative abundance of microbiome composition between aposymbiotic and symbiotic states in strain CC7 (Curtis et al., 2023;Röthig et al., 2016;Sydnor, 2020).Nevertheless, the four differentially abundant taxa we identified were not previously reported, suggesting genotype by environment and symbiotic state all influence microbial assemblages in Aiptasia.

| Microbiome fluctuations caused by heat stress changed the rare microbiome
Here, we aimed to identify microbial taxa that are linked with symbiotic states in a defined clonal strain of Aiptasia and tracked microbial fluctuations of these taxa following mild bleaching.The dominant taxa in all Aiptasia were found to be unaffected by both temperature and symbiotic state.However, the rare microbiome of symbiotic Aiptasia was significantly affected by temperature stress.In contrast to the other treatment groups, symbiotic Aiptasia exhibited a reduction in alpha-diversity and beta-diversity convergence after 6 days of exposure to heat stress (Figure 3).A decrease in alpha diversity was also observed in another study exposing symbiotic Aiptasia CC7 to short-term heat stress (Sydnor, 2020), but it appears to be a temporary phenomenon since a long-term study by Ahmed et al. (2019) on Aiptasia CC7, surveying microbial communities under continuous heat stress (32°C for 2 years), demonstrated an increase in both alpha diversity and number of bacterial taxa relative to paired controls.Here, symbiotic Aiptasia experienced microbiome restructuring when subjected to heat stress (Figure 4d) and the resulting assemblage was most similar to that observed in heat-stressed aposymbiotic anemones (Figure 4c).This suggests that short-term heat stress in Aiptasia may be the main driver that led to a convergence of microbial communities in both aposymbiotic and symbiotic animals.However, possible symbiont gains in the aposymbiotic and symbiont loss in the symbiotic anemones exposed to thermal stress (Figure S3) may also be contributing to the convergence of microbial communities.Symbiotic anemones exhibited increased relative abundance of several bacteria taxa, most noticeably Oligoflexales, Saccharospirillaceae, and Pseudoalteromonadaceae compared to aposymbiotic anemones under control temperatures (Figure 4b).These same taxa increase in relative abundance in heat-stressed aposymbiotic anemones compared to control aposymbiotic anemones (Figure 4a) until their relative abundance is indistinguishable from those observed in the symbiotic anemones in response to thermal stress (Figure 4c), whereas their abundances decrease in response to heat stress in symbiotic anemones (Figure 4d).The most parsimonious explanation for these apparently contradictory changes in response to heat stress is that it is not thermal stress, but algal symbiont density which influences patterns of convergence.Additional time course studies examining repopulation of algal symbiont communities would provide additional support for this hypothesis.
It is unclear whether changes in the relative abundance or composition of the microbial community affected the physiology or fitness of aposymbiotic and symbiotic Aiptasia hosts as we did not conduct any host-specific assays, but we can confirm that no Aiptasia died during the course of this experiment.While we have identified a strong association between the abundance of Oligoflexales and algal endosymbionts independent of thermal stress, whether these bacteria are mutualists or commensals remains unresolved.Additional work on patterns of localization, metabolic exchange, and spatial and temporal fidelity are needed.But given the tractability of the Aiptasia model, this represents a promising future study system for investigating multipartner symbioses.Understanding how the absence of a member in a multipartite symbiosis impacts the resilience of other organisms in the holobiont can uncover valuable insights into how symbiotic organisms respond to environmental challenges.

Firmicute
abundances were higher in aposymbiotic and symbiotic controls (Figure 4a,d), and Oligoflexales abundances were highest in symbiotic anemones under control conditions (Figures 2 and 4b ).Bacteria from the family Methylophilaceae exhibited a similar pattern as Oligoflexales, but overall abundance was low, and the difference between aposymbiotic and symbiotic controls was only ~100 raw counts.In contrast, Oligoflexales abundances in the aposymbiotic and symbiotic controls differed by 1-3 orders of magnitude (Figure5b).Alteromonadales and Oceanospirillales abundance both decreased in heat-stressed anemones, regardless of symbiotic state (Figure4c).

F
Relative abundance barplots of taxa, by class, in aposymbiotic and symbiotic anemones by experimental conditions (25°C vs. 32°C).Alphaproteobacteria, Bdellovibrionota, and Gammaproteobacteria dominate all the samples.Oligoflexia are distinctly present in control symbiotic anemones.Individual replicates have been collapsed by treatment.F I G U R E 3 Alpha-diversity and beta-diversity differences of microbial assemblages in Aiptasia.(a) Alpha-diversity index, Chao1, by treatment group.Standard error for Chao1 is represented by the error bars.(b) Beta differences by weighted Unifrac, principal coordinates of analysis (PCoA) on aposymbiotic samples (control, dark blue circles vs. heat stressed, light blue circles), and symbiotic (control, dark brown triangles vs. heat stressed, light brown triangles).

F
I G U R E 5 Oligoflexales in the microbiome of Aiptasia.(a) Regression line through the origin and linear model results testing the relationship between the dependent variable, Oligoflexales bacterial counts (y-axis) and explanatory variable, symbiont-to-host ratio (S/H) (x-axis).(b) Total counts of Oligoflexales in rarefied data, by treatment group (black circles = aposymbiotic anemones, black triangles = symbiotic anemones).Inset in Figure 5b shows a magnified view of low abundance Oligoflexales counts in the aposymbiotic control anemones (y-axis = 0-20 counts).